Process for producing pyridoxal-5-phosphate



United States Patent ABSTRACT OF THE DISCLOSURE Pyridoxal-S-phosphate isobtained by reacting 4-methyl- 5-lower alkoxyoxazole with 2,5-di-loweralkoxy-2,5-dihydrofuran to form an adduct, treating the compound with analkali metal hydroxide to form l,3-dihydro-l,3-di-loweralkoxy-fi-methylfuro[3,4-c1pyridin-7-ol, hydrolyzing the product with anacid to 2-methyl-3-hydroxypyridine-4,5- dicarboxaldehyde, reacting theproduct with 2-cyclohexylaminoethanethiol or 2-benzylaminoethanethiol toafford 2-(3-hydroxy-5-forn1yl-2-methyl-4-pyridyl)-3cyclohexylthiazolidine or 2-(3-hydroxy-S-formyl-Z-methyl 4pyridyl)-3-benzylthiazolidine, respectively, reducing the product to 2(3 hydroxy-S-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidineor 2-(3-hdroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-benzylthiazolidine, respectively, phosphorylating the product with a mixtureof orthophosphoric acid and phosphorus pentoxide, heating the reactionmixture with the addition of water, and then treating the resultantmixture with an alkali metal hydroxide.

The present invention relates to a novel process for the manufacture ofpyridoxal-S-phosphate and also to novel intermediates utilized and/ orformed in the above-described process.

Pyridoxal-S-phosphate is a very useful substance as a medicine, and anumber of processes have hitherto been known for manufacturing the same,for example, by oxidation or transamination of pyridoxamine-S-phosphate[E. A. Peterson et al., Journal of the American Chemical Society, vol.76, 169 (1954) and R. F. Long, British Patent No. 749,800] or byphosphorylation of pyridoxal after protection of its aldehyde group andby subsequent removal of the protecting group [P. Karrer et al.,Helvetica Chimica Acta, vol. 34, 1834 (1951); G. Schorre, US. Patent No.3,124,587 and Murakami et al., Japanese Patent No. 472,627].

These known processes invariably depend on pyridoxal or pyridoxamine asa starting material. As is known to the art, pyridoxal and pyridoxamineare costly because both are prepared from pyridoxine. Thus, thepyridoxal-S-phosphate prepared by any of the known processes isnaturally high-priced.

It is therefore an object of the present invention to provide a processfor economically manufacturing pyridoxal-S-phosphate.

Another object of the invention is to provide a process formanufacturing pyridoxal-S-phosphate from inexpensive materials, withoutusing any expensive one such as pyridoxine, pyridoxal or pyridoxamine.

The process of the invention can be expressed by the following reactionscheme:

CH3 0 H3 (III) (Iv) (l /HO HO GHO Ra-NH--CH2CH;SH

(V) VI Y a s N-R Ho H0 CH OH 2 phosphorylation CH3 N N VII (VIII)reaction product H0 C H2O P 0 H,

wherein R and R may be the same or difierent and represent lower alkylgroups having from one to five carbon atoms and R represents acyclohexyl or benzyl group.

The process of the invention comprises the steps of reacting4-methyl-5-lower alkoxyoxazole (I) with 2,5-diloweralkoxy-2,5-dihydrofuran (II) to form an adduct (III), treating theadduct with an alkali metal hydroxide to form l,3-dihydro-l,3-di-loweralkoxy-6-methylfuro[3,- 4-cJ-pyridin-7-ol (IV), hydrolyzing the productwith an acid to 2-methyl-3-hydroxypyridine-4,S-dicarboxaldehyde (V),reacting the product with 2-cyclohexylaminoethanethiol orZ-benzylaminoethanethiol (VI) to afford2-(3-hydroxy-S-formyl-2methyl-4-pyridyl)-3 cyclohexylthiazolidine or2-(3-hydroxy-5-formyl-2-methyl-4-pyridyl)3-beuzylthiazolidine (VII),respectively, reducing the product to 2(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridy1)-3- cyclohexylthiazolidineor 2(3-hydroxy-5-hydroxmethyl-2- methyl-4-pyridyl) -3-benzylthiazolidine(VIII), respectivel, phosphorylating the product with a mixture oforthophosphoric acid and phosphorus pentoxide, heating the reactionmixture with the addition of water, and then treating the mixture withan alkali metal hydroxide, thereby to obtain desiredpyridoxal-S-phosphate (X).

As briefly described above, the present inventors have surprisinglydiscovered the following unexpected facts.

First, the new compound (V) can be synthesized in a good yield in asimple manner starting from the reaction of (I) with (II), although thepreparation of a pyridine derivative having two vicinal aldehyde groupshas generally been considered troublesome.

Secondly, the reaction of Z-methyl-3-hydroxypyridine-4,5-dicarboxaldehyde (V) with 2-cyclohexyla-minoethanethiol or2-benzylarninoethanethiol (VI) gives exclusively the4-thiazolidinyl-S-formylpyridine derivative (VII) in a nearlyquantitative yield Without formation of any by-product, and thisselective reaction takes place only with 2-cyclohexylaminoethanethioland 2-benzylaminoethanethiol among various aminoethanethiol derivativesinvestigated. For example, the reaction of (V) with aminoethanethiolgives no thiazolidine derivative but a resinous product. Also, thereaction of (V) with Z-methylaminoethanethiol affords a considerableamount of byproduct (mainly the 4,5-dithiazolidinylpyridine derivative)in addition to the 4-thiazolidinyl-S-formylpyridine derivative. Further,in the reaction of (V) with Z-pheny aminoethanethiol, the4-thiazolidinyl-5-formylpyridine derivative is obtained in only about 20percent yield. Thus, the use of either 2-cyclohexylaminoethanethiol or2-benzylaminoethanethiol brings about an unexpectedly high yield offormation of the 4-thiazolidinyl-S-formylpyridine derivative (VII) andmakes the present invention to be an advantageous method for theproduction of pyridoxal- 5-phosphate.

Thirdly, the aldehyde group at the 5-position of a compound of theFormula VII is readily reduced to give a compound of the Formula VIII inan excellent yield.

The present invention has been completed on the basis of the abovefindings. According to the process of the invention,pyridoxal-S-phosphate is advantageously manufactured via novelintermediates of the Formulas III, IV, V, VII and VIII.

One of the starting materials for the process of the invention is acompound of the Formula I wherein R is a straightor branched-chain loweralkyl group such as methyl, ethyl, propyl, isopropyl, or butyl group andis readily prepared according to the method described by Harris et al.in Journal of Organic Chemistry, vol. 27, 2705 (1962). The otherstarting material for the process of the present invention is a compoundof the Formula II wherein R is a straightor branched-chain lower alkylgroup such as methyl, ethyl, propyl, isopropyl, or butyl group, and caneasily be obtained according to the method described by Clauson-Kaas etal. in Acta Chemica Scandinavica, vol. 6, 531 (1952) or by Hufford etal. in Journal of the American Chemical Society, vol. 74, 3014 (1952).The compound of the Formula II prepared by the above methaods is amixture of cis and trans stereoisomers, both of which may be isolated bydistillation. In the process of the present invention either cis ortrans isomer can be used, although the cis isomer has been proved totake part in the reaction with (I) more easily than the trans isomer. Inthe process of the present invention, (II) is advantageously used as amixture of cis and trans isomers, and the products (III) and (IV)obtained in the subsequent steps may be a mixture of cis and transisomers with respect to the two alkoxy groups. It is to be noted thatthe process of the present invention can be carried out without respectto the stereoisomerism mentioned above.

The reaction of (I) with (II) for the production of (III) proceeds atroom temperature but is preferably carried out at a temperature between50 and 200 C., especially between 100 and 150 C. The use of solvent isnot essential, but a conventional organic so vent such as ethanol,benzene, toluene or xylene may be employed. In the reaction, a smallamount of hydroquinone can be effectively added to prevent sidereactiions such as polymeria t n- It is al o desi ab e to ca y out the ra tion n an atmosphere of inert gas such as nitrogen or argon. It ispossible to carry out the reaction using one mole of (II) per mole of(I), but it is generally preferable to use (II) in excess, for example,3 to 15 moles per mole of (I). Thus, (III) can be obtained in a goodyield by reacting (I) with (II) at a temperature between 100 and 150 C.for a period of one to 15 hours.

Concentration in vacuo of the reaction mixture thus obtained gives anadduct of the Formula III as a residue, and a mixture of the unreactedstarting materials (I) and (II) is recovered as a distillate. Theresidue may contain theoretically eight kinds of stereoisomers withrespect to the endo-exo adductation and the cis-trans isomerism ofdialkoxy groups on the tetrahydrofuran ring. In the case in which R isan ethyl group and R is a methyl group, recrystallization of the residuefrom ligroin affords a compound of melting point 113 C. and purificationof the mother liquor by alumina chromatography followed byrecrystallization from petroleum benzine gives another compound ofmelting point C. On the basis of elemental analysis and spectral data,it is assumed that the former compound is an endo-adduct and the latteran exoadduct and that both compounds have cis dimethoxy groupscorresponding to the Formula III. In practicing the process of theinvention, however, it is not necessary to isolate these crystallinesubstances, but the crude product obtained above by concentrating thereaction mixture may be used in the next step.

The unreacted starting materials (I) and (II) are efficiently recoveredas a distillate by concentration of the reaction mixture and can bere-used after complementary additon of (I) and (II). Accordingly, byrepeating the above procedure, the adduct (111) can be obtained in ahigh yield.

The adduct (III), when treated with an alkali metal hydroxide such assodium hydroxide or potassium hydroxide, can readily be converted to acompound of the Formula IV. The reaction is smoothly effected in asuitable solvent such as water, methanol, ethanol, dioxane or a mixturethereof at a temperature between room temperature and 100 C. It isparticularly desirable to treat (III) with sodium or potassium hydroxidein a lower aliphatic alcohol such as methanol or ethanol at atemperature between 60 and C. By this procedure, (IV) is obtained in asubstantially quantiative yield. In the where R is a methyl group,recrystallization of the crude product from methanol affords a compoundof melting point 165166 C. corresponding to the Formula IV which hasbeen shown by NMR (nuclear magnetic resonance) spectral analysis to havecisdimethoxy groups on the dihydrofuran ring.

Next, (IV) is hydrolyzed with an acid such as hydroch oric acid,sulfuric acid, or acetic acid in water, or in a mixture of water and anorganic solvent miscible with water such as methanol, ethanol or dioxaneat a temperature between room temperature and C. In this way, a compoundof the Formula V is quantitatively formed, but the product is in generalisolated as a cyclic monohydrate of (V), that is,1,3-dihydro-1,3-dihydroxy-6-methylfuro 3, 4-c pyridin-7-ol which isshown by the following formula:

The NMR spectral analysis shows the existence of an equilibrium betweenthe dialdehyde structure (V) and its cyclic monohydrate In practicingthe process of the invention, however, isolation of the product is notnecessary and the reaction mixture obtained by hydrolysis canconveniently be employed in the subsequent step.

The reaction of (V) with (VI) is carried out in a solvent such as water,an organic solvent, e.g., methanol,

ethanol, or dioxane or a mixture thereof at a temperature between roomtemperature and 100 C. Both compounds (V) and (VI) may be used as eitherfree bases or acid salts and are conveniently employed in substantiallyequimolar amounts. In the case where free bases of (V) and (VI) areused, the reaction proceeds smoothly at a temperature between roomtemperature and 100 C. When either or both of (V) and (VI) are used asacid salts, they are liberated by neutralization with an alkali metalhydroxide, carbonate or hydrogen carbonate before or after the two arebrought into contact with each other. In this way, the compound (VII) isformed in an excellent yield and can be isolated in a pure state. In thepractice of the process of the invention, however, the compound (VII),without being isolated, can advantageously be subjected to the nextreaction.

In the reduction of (VII) to (VIII), conventional methods can beemployed. For example, the reduction of (VII) with sodium borohydride,aluminum isopropoxide in isopropanol or zinc powder in an aqueous aceticacid afiords (VIII) in an excellent yield.

In the final phosphorylation step, a mixture of orthophosphoric acid andphosphorus pentoxide is used as a phosphorylating agent. The reagent wasdescribed by R. E. Ferrell et al. in Journal of the American ChemicalSociety, vol. 70, 2101 (1948). Thus, the compound (VIII) is easilyphosphorylated when heated with stirring with this reagent at atemperature between 40 and 80 C. for one to hours. The product (IX)resulting from the phosphorylation is presumed to be a compound whichmay be expressed by the following formula:

HO ]CHgOR4 CH3 wherein R represents a polyphosphoric acid group and Rrepresents the same as defined above. The resultant product (IX),without being isolated, is converted to (X) by hydrolysis of both thepolyphosphoric acid group and the thiazolidine ring. The reaction iscarried out by heating the reaction mixture with the addition of waterto first convert the polyphosphoric acid group to a monophosphoric acidgroup and subsequent treatment with an alkali metal hydroxide to thenhydrolyze the thiazolidine ring. Thus, the above-mentionedphosphorylation mixture is heated with the addition of water at atemperature between 90 and 100 C. for 30 minutes to one hour, and thenis treated at room temperature with an alkali metal hydroxide such assodium or potassium hydroxide to adjust the pH of the solution to 10 ormore.

For the isolation of the final product (X), the solution resulting fromthe above hydrolysis can be purified using, for example, active carbonor an ion-exchange resin such as Am berlite CG-50 or Amberlite IR-120(trademarks of Rhom & Haas Co.). In the process of the invention,however, a preferably procedure is to add an organic base such as4-aminoantipyrine, a-naphthylamine or ,B-naphthylamine to the solutionresulting from the above hydrolysis, neutralize the solution with anacid such as hydrochloric or acetic acid to precipitate the product (X)as a Schitf base, isolate the Schiff base, decompose it with an aqueoussolution of an alkali metal hydroxide such as sodium or potassiumhydroxide, remove the liberated organic base from the solution, treatthe solution with an ion-exchange resin or neutralize with an acid, andthen evaporate the resultant solution, thus affordingpyridoxal-S-phosphate of high purity in a good yield.

The following examples are given by way of illustration only and are notto be construed as limiting.

EXAMPLE 1 A mixture of 5 g. of 4-methyl-5-ethoxyoxazole and 50 g. of2,5-dimethoxy-2,S-dihydrofuran was heated at 130 C. under nitrogen in asealed tube for 8 hours. The reaction mixture was distilled underreduced pressure, 47 g. of a mixture of the unreacted4-methyl-5-ethoxyoxazole and 2,5-dimethoxy-2,S-dihydrofuran wasrecovered as a distillate and 7.0 g. of a crude adduct was obtained as ahalf-crystalline residue. Part of the residue was dissolved in benzene,passed through a column of alumina and, after evaporation of thesolvent, recrystallized from ligroin to give colorless crystals of theendo-adduct (III), M.P. 113 C.

Analysis.Calcd. for C H O N (percent): C, 56.02; H, 7.44; N, 5.44. Found(percent): C, 56.04; H, 7.54; N, 5.5.

The mother liquor obtained by separation of the endoadduct wasevaporated, the residue in petroleum benzine was chromatographed onalumina and eluted with petroleum benzine with increasing amount ofbenzene. From the petroleum benZine-benzene (9:1 and 1:1) eluate anotherproduct of M.P. 7 C. was obtained after recrystallization from petroleumbenzine, which corresponds to an exo-adduct (III).

Analysis.Calcd. for C H O N (percent): C, 56.02; H, 7.44; N, 5.44. Found(percent): C, 55.65; H, 7.46; N, 5.2.

Three grams of adduct, M.P. 113 C., obtained above was heated underreflux in 60 ml. of a 5% potassium hydroxide methanol solution for 6hours. The reaction mixture was neutralized with concentratedhydrochloric acid and an inorganic precipitate was separated byfiltration. The filtrate was concentrated to give a crystalline residue,which was extracted with chloroform. Evaporation of the extract afforded2.3 g. of 1,3-dihydro-1,34iimethoxy-6-rnethylfuro[3,4-c]pyridin-7-ol. Apure sample of M.P. -166 C. was obtained by recrystallization frommethanol.

Analysis.-Calcd. for C H O N (percent): C, 56.86; H, 6.20; N, 6.63.Found (percent): C, 57.21; H, 6.17; N, 6.74.

In the same manner as described for the adduct of M.P. 113 C., theadduct of M.P. 75 C. obtained above was treated with a potassiumhydroxide methanol solution to give the same compound,1,3-dihydro-1,3-dimethoxy-6-methylfuro[3,4-c]pyridin-7-ol, M.P. 165 C.

A solution of 0.42 g. of l,3-dihydro-1,3-dimethoxy-6-methylfuro-[3,4-c1pyridin-7-ol obtained above in 5 ml. of 10%hydrochloric acid was heated at 100 C. for 10 minutes. The reactionmixture was concentrated at a low temperature under reduced pressure togive a colorless crystalline residue of2-methyl-3-hydroxypyridine-4,5-dicarboxaldehyde monohydratehydrochloride, i.e., 1,3-dihydr0-1,3-dihydroxy-6-methylfuro 3,4-c]pyridin-7-ol hydrochloride, which exhibited no definite melting point.Ultraviolet absorption maximum: 288rn in normal hydrochloric acid.

Analysis.Calcd. for C H O N-HCl: C, 43.74; H, 4.59; N, 6.38; Cl, 16.14.Found: C, 43.98; H, 4.62; N, 6.37; Cl, 16.48%.

0.5 g. of 2-methyl-3-hydroxypyridine-4,5-dicarboxaldehyde monohydratehydrochloride obtained above was dissolved in minimum amount of water,neutralized with icecooling to pH 7 using a normal sodium hydroxidesolution and the precipitate was separated by centrifuge, washed withcold water. The product was recrystallized from a small amount of waterand dried under reduced pressure at a temperature lower than 50 C. toafford a white crystalline solid which corresponds to 1,3-dihydro-1,3-dihydroxy-6-methylfuro[3,4-c]pyridin-7-ol having 1.5 mols of waterof crystallization. It had no clear melting point.

Analysis.Calcd. for C H O N-1.5H O: C, 45.71; H, 5.75; N, 6.66. Found:C, 45.16; H, 5.23; N, 6.29%.

0.46 g. of 2-methyl-3-hydroxypyridine-4,S-dicarboxaldehyde monohydratehydrochloride obtained above was dissolved in 6 ml. of water and to thissolution was added 0.32 g. of 2-cyclohexylaminoethanthiol in 3 ml. of 1N hydrochloric acid. The solution was heated to 4050 C. and adjusted topH 78 with a concentrated sodium hydroxide solution. An oily materialwas separated and extracted with chloroform. The extract was dried overanhydrous sodium sulphate and evaporated to give a crystalline residue.Recrystallization of the residue from acetone afforded 2- (3-hydroxy--formyl-2-methyl-4-pyridyl) -3 -cyclohexylthiazolidine, M.P.107-109" C.

Analysis.- Calcd. for C H O N S: C, 62.71; H, 7.24; N, 9.14; S, 10.46.Found: C, 62.60; H, 7.22; N, 8.90; S, 10.17%.

A solution of 0.42 g. of 2-(3-hydroxy-5-formyl-2-rnethyl-4-pyridyl)-3-cyclohexylthiazolidine obtained above in aqueousmethanol was treated with 0.07 g. of sodium borohydride and allowed tostand at room temperature for one hour. Evaporation of methanol from thereaction mixture quantitatively precipitated 2-(3-hydroxy-5-hydroxymethyl 2-n1ethyl 4-pyridyl) 3 cyclohexylthiazolidine, whichshowed M.P. 198200 C. Recrystallization from acetone raised the M.P. to199200.5 C.

AnaIysis.-Calcd. for C H O N S: C, 62.30; H, 7.84; N, 9.08; S, 10.40.Found: C, 62.69; H, 8.04; N, 8.90; S, 10.26%.

0.62 g. of 2-(3-hydroxy-5-hydroxymethyl-2-n1ethyl-4-pyridyl)-3-cyclohexylthiasolidine thus obtained was added to a mixtureof 3.0 g. of 85% orthophosphoric acid and 2.5 g. of phosphorus pentoxideand the mixture was heated with stirring at 60 C. for 5 hours. Aftercooling, the reaction mixture was treated with 20 ml. of water andheated at 100 C. for 30 minutes. The solution was cooled to roomtemperature and added with 3.0 g. of active carbon to adsorb theproduct. The active carbon was thoroughly washed with water and elutedwith 200 ml. of 1 N sodium hydroxide. The eluate was concentrated to asmall volume under reduced pressure, poured onto a column (3.2 x 60 cm.)of acidic ion-exchange resin, Amberlite CG50, and eluted with water.Fractions containing pyridoxal-S-phosphate were collected, concentratedto a small volume at a low temperature under reduced pressure andallowed to stand in a refrigerator. White crystals precipitated werecollected by filtration to give 0.32 g. of pyridoxal-S-phosphatemonohydrate, which was identical in all respects with an authenticsample of pyridoxal-S-phosphate monohydrate.

EXAMPLE 2 A solution of 2.6 g. of 4-methyl-5-ethoxyoxazole and 26 g. of2,5-dimethoxy-2,S-dihydrofuran was heated at 100 C. for 20 hours undernitrogen in a sealed tube. The reaction mixture was concentrated underreduced pressure to give 3.0 g. of an adduct as a residue and a mixtureof the unreacted 4-methyl-5-ethoxyoxazole and2,5-dimethoxy-2,S-dihydrofuran as a distillate. The adduct was disolvedin 60 ml. of a 5% potassium hydroxide methanol solution and the solutionwas heated under reflux for 40 hours. After cooling, the reactionmixture was neutralized with concentrated hydrochloric acid to separateinorganic material, which Was filtered off. The filtrate was evaporatedto give a crystalline residue, which was extracted with chloroform. Theextract was concentrated to afford 1.96 g. of1,3-dihydro-1,3-dimethoxy-6-rnethylfuro [3 ,4-c] pyridin-7-ol.

2.8 g. of 1,3-dihydro-1,3-dimethoxy-6-methylfuro[3,4- c]-pyridin-7-olthus obtained was dissolved in 40 ml. of 10% hydrochloric acid and thesolution was heated at 100 C. for 10 minutes. The solution was cooled,adjusted to pH 78 with a concentrated sodium hydroxide solution, addedwith 80 ml. of methanol and the mixture was heated to 40-50 C., addedwith 2.22 g. of 2-cyclohexylaminoethanethiol in 10 ml. of methanol,maintained at this temperature for 30 minutes. Then, the mixture wascooled to room temperature, treated with 0.25 g. of sodium borohydrideand allowed to stand at room ternperature for one hour. Evaporation ofmethanol from the reaction mixture gave a precipitate, which wasfiltered to give 3.38 g. of 2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidine, M.P. 199200 C.

0.62 g. of 2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidine obtained above was agitated with amixture of 3.0 g. of orthophosphoric acid and 2.5 g. of phosphoruspentoxide at 50 C. for 3 hours. 20 ml. of water was added to thereaction mixture and the solution was heated at C. for 30 minutes,cooled, and made alkaline with the addition of 8 g. of sodium hydroxidein 40 m1. of water. The solution was treated with 0.40 g. of4-amino-antipyrine and the pH of the solution was adjusted to 3 withconcentrated hydrochloric acid, and then the mixture was allowed tostand overnight in a refrigerator. The separated Schiff base wascollected by filtration, washed with water, dissolved in 10 ml. of 2 Nsodium hydroxide, and extracted with benzene to remove 4-amino-antipyrine formed. The aqueous layer was neutralized withhydrochloric acid, concentrated to a small volume under reduced pressureat a low temperature and allowed to stand overnight in a refrigerator.The separated crystals were collected by filtration to give 0.28 g. ofpyridoxal-S-phosphate monohydrate.

EXAMPLE 3 A solution of 2.54 g. of 4-methyl-5-ethoxyoxazole and 26 g. of2,5-dimethoxy-2,S-dihydrofuran was heated at C. for 4 hours undernitrogen in a sealed tube. The reaction mixture was concentrated underreduced pressure to give an adduct as a residue and a mixture of theunreacted 4-.methyl-5-ethoxyoxazole and 2,5-dimethoxy-2,5- dihydrofuranwas recovered as a distillate. To the distillate which contains about50% of 4-methyl-5-ethoxyoxazole used was added 1.27 g. of4-methyl-5-ethoxyoxazole and the solution was adjusted to the originalmolar ratio by addition of appropriate amount of 2,5-dimethoxy-2,5-dihydrofuran and again heated at 130 C. for 4 hours undernitrogen in a sealed tube. This reaction procedure was repeated tentimes, giving 24.5 g. of an adduct, The product was dissolved in 500 ml.of a 5% potassium hydroxide methanol solution and the mixture was heatedunder reflux for 5 hours. After cooling, the reaction mixture wasneutralized with concentrated hydrochloric acid, the precipitatedinorganic material was separated by filtration and the filtrate wasconcentrated under reduced pressure to give 1,3-dihydro-1,3-dimethoxy-6-methylfuro[3,4-c]pyridin-7-ol as a crystalline mass.

The product obtained above was dissolved in 250 ml. of 10% hydrochloricacid and the solution was heated at 100 C. for 10 minutes, neutralizedwith ice-cooling to pH 78 with a concentrated sodium hydroxide solution,added with 300 ml. of methanol, heated again to 40-50 C., treated with asolution of 14.5 g. of 2-cyclohexylaminoethanethiol in 20 ml. ofmethanol and maintained at this temperature for 30 minutes. To thismixture was added 1.70 g. of sodium borohydride at room temperature andthe solution was agitated for one hour. Evaporation of methanol from thereaction mixture afforded a precipitate, which was separated byfiltration to give 23.1 g. of2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidine, M.P. 196198 C.

0.62 g. of 2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidine obtained above was agitated with amixture of 3.0 g. of 85 orthophosphoric acid and 2.5 g. of phosphoruspentoxide at 50 C. for 5 hours and the solution was treated with 20 ml.of water, heated at 100 C. for 30 minutes. The reaction mixture waspoured into a solution of 8 g. of sodium hydroxide in 40 ml. of Water tobe rendered alkaline, treated with 0.40 g. of 4-aminoantipyrine,adjusted to pH 3 with hydrochloric acid and allowed to stand overnightin a refrigerator. The precipitated Schiff base was separated byfiltration, washed with water and dissolved in 10* ml. of 2 N sodiumhydroxide. The mixture was extracted with benzene to remove4-aminoantipyrine formed and the aqueous layer was poured onto a columnof an ion-exchange resin, Arnberlite CG-SO, and eluted with water.Fractions containing pyridoxal-S-phosphate were collected, concentratedto a small volume under reduced pressure at a low temperature andallowed to stand overnight in a refrigerator. The precipitate wasfiltered to give 0.37 g. of pyridoxal-S-phosphate monohydrate.

EXAMPLE 4 A mixture of 0.64 g. of 4-.methyl-S-ethoxyoxazole, 6.5 g. of2,5-dimethoxy-2,5-dihydrofuran and 20 mg. of hydroquinone in 10 ml. ofbenzene was heated under reflux for 40 hours. Concentration of thereaction mixture gave 060 g. of an adduct as a half-crystalline residue.The residue was treated in the same manner as in Example 1 to give 1,3dihydro-1,3 dimethoxy 6 methylfuro [3,4-c] pyridin-7-ol.

0.42 g. of 1,3-dihydro-1,3-dimethoxy-6-methylfuro[3,4- c]-pyridine-7-olobtained was dissolved in ml. of hydrochloric acid and heated at 100 C.for 10 minutes. The reaction mixture was cooled, adjusted to pH 78 witha concentrated sodium hydroxide solution, added with 0.32 g. of2-cyclohexylaminoethanethiol and heated at 5060 C. for 10 minutes. Theseparated oil was extracted with chloroform and the extract was driedover anhydrous sodium sulphate and evaporated to give 2-(3- hydroxy 5formyl 2 methyl 4 pyridyl) 3 cyclohexylthiazolidine as a crystallineresidue. The product was dissolved in 50 ml. of isopropanol and heatedunder reflux for 24 hours with 0.61 g. of aluminum isopropoxide. Theresidue obtained by evaporation of the solvent was treated with waterand the mixture was acidified to pH 2 with hydrochloric acid and thenneutralized to pH 7 with a sodium carbonate solution. The mixture wasextracted with chloroform and the extract after drying over anhydroussodium sulphate was evaporated to afford 0.41 g. of2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)3-cyclohexylthiazolidine as a crystalline residue, M.P. 199- 200 C.

In the same manner as described in Example 3, 0.62 g. of2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidine obtained above was phosphorylated andhydrolyzed to give 0.37 g. of pyridoxal-S-phosphate monohydrate.

EXAMPLE 5 A mixture of 2.6 g. of 4-methyl-5-ethoxyoxazole and 37 g. of2,5-diisopropoxy-2,5-dihydrofuran was heated at 100 C. for hours undernitrogen in a sealed tube. The reaction mixture was concentrated underreduced pressure to give a crude adduct as a residue and a mixture ofthe unreacted 4-methyl-5-ethoxyoxazole and 2,5-diisopropoxy-2,5-dihydrofuran was recovered. The adduct was dissolved ina 5% potassium hydroxide methanol solution and the mixture was heatedunder reflux for 5 hours. After cooling, the reaction mixture wasneutralized with a concentrated hydrochloric acid to separate aninorganic material, which was filtered off. The filtrate wasconcentrated to dryness in vacuo and the residue was extracted withchloroform. The extract was evaporated to give 1.25 g. of1,3-dihydro-1,3-diisopropoxy-6-methylfuro [3,4-c]pyridin-7-ol which,after recrystallization from acetone, showed M.P. 153154 C.

Analysis.Calcd. for C H O N: C, 62.90; H, 7.92; N, 5.24. Found: C,62.64; H, 7.85; N, 5.29 percent. 7

0.50 g. of 1,3-dihydro-1,3-diisopropoxy-6-methyl-furo[3,4-c]-pyridin-7-ol obtained above was dissolved in 6 ml. of 10%hydrochloric acid and the solution was heated at 100 C. for 10 minutes.The reaction mixture was concentrated to dryness under reduced pressureto give 2- 10 methyl-3-hydroxypyridine-4,5-dicarboxaldehyde monohydratehydrochloride as a colorless crystalline residue.

In the same manner as described in Example 1, 2-methyl-3-hydroxypyridine-4,5-dicarboxaldehyde monohydrate hydrochlorideobtained above was treated to give pyridoxal-S-phosphate monohydrate.

EXAMPLE 6 In the same manner as described in Example 1, 1,3-dihydro-1,3-dimethoxy-6-methylfuro [3 ,4-c] pyridin-7-ol was prepared. 0.42 g. ofthe above-obtained product was dissolved in 5 ml. of 10% hydrochloricacid and heated at 100 C. for 10 minutes. The reaction mixture wascooled, adjusted to pH 7-8 with a sodium hydroxide solution and warmedat 50-60 C. for 10 minutes after addition of 0.32 g. of2-cyclohexylaminoethanethiol. To the cooled reaction mixture were added3 ml. of glacial acetic acid and 0.15 g. of zinc powder and the mixturewas agitated at 5-10" C. for one hour. The reaction mixture wasevaporated to dryness under reduced pressure and the residue was treatedwith water and extracted with chloroform. The chloroform extract afterwashing and drying over anhydrous sodium sulphate was evaporated toafford. a residue, which was recrystallized from acetone to give 0.20 g.of 2-(3-hydroxy5-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidine.

0.62 g. of 2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-cyclohexylthiazolidine obtained above was agitated at 50 C.for 5 hours with a mixture of 3.0 g. of orthophosphoric acid and 2.5 g.of phosphorus pentoxide. 20 ml. of water was added with cooling to thereaction mixture and the solution was heated at C. for 30 minutes,cooled and made alkaline by addition of 8 g. of sodium hydroxide in 40ml. of water. 0.29 g. of a-napthylamine was added to the solution, thepH of the solution was brought to 3 with hydrochloric acid and themixture was allowed to stand overnight in a refrigerator to give aSchiff base as a precipitate. The precipitate was worked up in the sameway as in Example 3 to give 0.37 g. of pyridoxal-S-phosphatemonohydrate.

EXAMPLE 7 In the same manner as described in Example 1, 2-(3-hydroxy-S-hydroxymethyl 2 methyl-4-pyridyl)-3-cyclohexylthiazolidine wasprepared. 0.62 g. of the aboveobtained product was agitated at 60 C. for3 hours with a mixture of 3.0 g. of 85 orthophosphoric acid and 2.5 g.of phosphorous pentoxide. The mixture was cooled, added with 10 ml. ofwater and rendered alkaline by treatment with 8 g. of sodium hydroxidein 40 ml. of water. The solution was poured onto a column (3.2 x 11 cm.)of Amberlite CG-50 and eluted with water. The eluate was added with anappropriate amount of concentrated hydrochloric acid to make thesolution have one normal hydrochloric acid concentration, heated at 80C. for 30 minutes and then treated with 3 g. of active carbon. Theactive carbon was thoroughly washed with water and eluted with 300 ml.of 1.5% ammoniacal water. The elute was concentrated to a small volumeunder reduced pressure, poured onto a column (3.2 x 60 cm.) of AmberliteCG-50 and eluted with water. Fractions containing pyridoxal-S-phosphatewere collected, concentrated to a small volume at a low temperatureunder reduced pressure and allowed to stand overnight in a refrigerator.The separated crystalline power was filtered to give 0.36 g. ofpyridoxal-S-phosphate monohydrate.

EXAMPLE 8 In the same manner as described in Example 1, 1,3-dihydro-,1,3 dimethoxy-G-methylfuro 3,4-c1pyridin-7-ol was prepared.2.11 g. of the above-obtained product was dissolved in 30 ml. of 10%hydrochloric acid and the solution was heated at 100 C. for 10 minutes,cooled and adjusted to pH 8 with a sodium hydroxide solution. Afteraddition of 50 ml. of methanol, the mixture was heated to 50 C., treatedwith 1.67 g. of Z-benzylaminoethanethiol and stirred for 30 minutes atthis temperature. Evaporation of methanol from the reaction mixtureseparated an oily material, which was extracted with chloroform. Theextract was passed through a column of small amount of alumina andevaporated to dryness to give a crystalline solid. The product wasrecrystallized from ethyl acetate to yield 2.4 g. of2-(3-hydroxy-5-formyl-2- methyl 4-pyridyl) 3 benzylthiazolidine, M.P.119.5- 120.5 C.

Analysis.-Calcd. for C H O N S: C, 64.94; H, 5.77; N, 8.91; S, 10.20.Found: C, 64.73; H, 6.01; N, 8.76; S, 10.16%.

To a solution of 0.31 g. of 2-(3-hydroxy-5-formyl-2-methyl-4-pyridyl)-3-benzylthiazolidine obtained above in 20 ml. of 50%aqueous methanol was added 0.07 g. of sodium borohydride and the mixturewas allowed to stand at room temperature for one hour. Evaporation ofmethanol from the reaction mixture afforded in quantitative yield 2- 3-hydroxy-S-hydroxymethyl-2-methyl-4-pyridyl) 3-benzylthiazolidine as acrystalline precipitate of M.P. l70172 C. A pure sample was obtained byrecrystallization from acetone, M.P. 171172 C.

AnaIysis.Calcd. for C H O N S: C, 64.53; H, 6.37; N, 8.85; S, 10.13.Found: C, 64.58; H, 6.27; N. 9.18; S, 9.91%.

0.63 g. of 2-(3-hydroxy-5-hydroxymethyl-2-methyl-4-pyridyl)-3-benzylthiazolidine obtained above was heated with stirring at50 C. for 4 hours with a mixture of 3.0 g. of 85 orthophosphoric acidand 2.5 g. of phosphorus pentoxide. 20 ml. of water was added to thereaction mixture and the solution was heated at 100 C. for 40 minutes,cooled and treated with 3.0 g. of active carbon. The carbon wasthoroughly washed with water and the product was eluted with 200 ml. of1 N sodium hydroxide. The eluate was concentrated to a small volumeunder reduced pressure, poured onto a column (3.2 x 60 cm.) of AmberliteCG-SO and eluted with water. Fractions containing pyridoxal-S-phosphatewere collected, concentrated to a small volume at a low temperatureunder reduced pressure and allowed to stand overnight in a refrigerator.The separated crystals were collected to give 0.31 g. ofpyridoxal-S-phosphate monohydrate. The product was identical with anauthentic sample of pyridoxal-S- phosphate monohydrate.

EXAMPLE 9 In the same manner as described in Example 1, 1,3- dihydro-1,3 dimethoxy-6-methylfuro[3,4-c1pyridin-7-ol was prepared. 2.11 g. ofthe above-obtained product was dissolved in 30 ml. of 10% hydrochloricacid and the solution was heated at 100 C. for 10 minutes, cooled andadjusted to pH 8 with a sodium hydroxide solution. After addition of 50ml. of methanol, the mixture was heated to 40-50 C., treated with 1.70g. of Z-benzylamineoethanethiol and stirred for 30 minutes at thistemperature. After cooling, 0.19 g. of sodium borohydride was added andthe mixture was allowed to stand at room temperature for 30 minutes.Evaporation of methanol from the reaction mixture afforded a crystallineprecipitate, which was filtered and washed with acetone to give 2.4 g.of 2-(3- hydroxy hydroxymethyl-2-methyl-4-pyridyl)-3-benzylthiazolidine,M.P. 168-170 C.

In the same manner as described in Example 8, 2-(3-droxy-S-hydroxymethyl-2-methyl-4 pyridyl)-3-benzylthiazolidine obtainedabove was phosphorylated to afford pyridoxal-S-phosphate monohydrate.

EXAMPLE 10 In the same manner as described in Example 8, 2-(3-hydroxy-5-hydroxymethyl-2-methyl 4 pyridyl)-3-benzylthiazolidine wasprepared. 0.63 g. of the above-obtained product was agitated with amixture of 3.0 g. of 85% orthophosphoric acid and 2.5 g. of phospohoruspentoxide at 50 C. for 3 hours. 20 ml. of water was added to thereaction mixture and the solution was heated at 100 C. for 30 minutes,cooled and made alkaline by addition of 8 g. of sodium hydroxide in 40ml. of water. Then, the mixture was treated with 0.40 g. of4-aminoantipyrine, adjusted to pH 3 with hydrochloric acid and allowedto stand overnight in a refrigerator. The separated Schiff base wascollected by filtration, washed with water and dissolved in 19 ml. of 2N sodium hydroxide. The mixture was extracted with benzene to remove 4-arninoantipyrine formed and the aqueous layer was neutralized withhydrochloric acid, concentrated to a small volume at a low temperatureunder reduced pressure and allowed to stand overnight in a refrigerator.Separated crystals were filtered to give 0.27 g. ofpyridoxal-S-phosphate monohydrate.

EXAMPLE 11 In the same manner as described in Example 8, 2-(3-hydroxy-S-hydroxymethyl 2-rnethyl-4-pyridyl -3 -'benzylthiazolidine wasprepared. 0.63 g. of the above-obtained product was heated with stirringwith a mixture of 3.0 g.

of orthophosphoric acid and 2.5 g. of phosphorus pentoxide at 60 C. for3 hours. 20 ml. of water was added to the reaction mixture and thesolution was heated at C. for 30 minutes, cooled and made alkaline byaddition of 8 g. of sodium hydroxide in 40 ml. of water. Then, themixture was treated with 0.29 g. of a-naphthylamine, adjusted to pH 3with hydrochloric acid and allowed to stand overnight in a refrigerator.The separated Schifr base was collected by filtration, washed with waterand dissolved in 10 ml. of 2 N sodium hydroxide. The mixture wasextracted with benzene to remove wnaphthylamine formed and the aqueouslayer was poured onto a column (3.2 x 60 cm.) of Amberlite CG50 andeluted with water. Fractions containing pyridoxal-S-phosphate werecollected, concentrated to a small volume at a low temperature underreduced pressure and allowed to stand overnight in a refrigerator.Separated crystals were collected by filtration to give 0.35 g. ofpyridoxal-S-phosphate monohydrate.

What is claimed is:

1. A process for the manufacture of pyridoxal-S-phosphate whichcomprises reacting 4-methyl-5-lower alkoxyoxazole of the generalformula:

one to five carbon atoms, with 2,5-di-lower alkoxy-2,5- dihydrofuran ofthe general formula:

wherein R represents a lower alkyl group having from one to five carbonatoms, to produce an adduct; treating the adduct with an alkali metalhydroxide to give 1,3- dihydro-1,3-di-loweralkoxy-6-methylfuro[3,4-c]-pyridin- 7-01 of the general formla:

wherein R represents the same as defined above; hydrolyzing the productwith an acid to 2-methyl-3-hydroxypyridine-4,S-dicarboxaldehyde;reacting the product with a compound of the general formula:

HO CHO CH3 \N wherein R represents the same as defined above; reducingthe product to the corresponding compound of the general formula:

wherein R represents the same as defined above; phosphorylating thecompound with a mixture of orthophosphoric acid and phosphoruspentoxide; heating the resultant reaction mixture with the addition ofwater; and finally treating the resultant mixture with an alkali metalhydroxide.

2. A process according to claim 1 wherein the 4-methyl- S-loweralkoxyoxazole used is 4-methyl-5-ethoxyoxazo1e.

3. A process according to claim 1 wherein the reaction of4-methyl-5-lower alkoxyoxazole with 2,5-di-lower alkoxy-2,5-dihydrofuranis carried out in an atmosphere of inert gas at a temperature between100 and 150 C.

4. A process according to claim 1 wherein the adduct is heated wtih analkali metal hydroxide selected from the group consisting of sodiumhydroxide and potassium hydroxide in a lower aliphatic alcohol at atemperature between 60 and 80 C. to give l,3-dihydro-l,3-di-loweralkoxy-o-methylfuro 3,4-c] pyridine-7-ol.

5. A process according to claim 1 wherein the hydrolysis ofl,3-dihydro-l,3-di-lower alkoxy-6-methylfuro- [3,4-c]pyridin-7-ol iscarried out in an aqueous solution of hydrochloric acid at a temperaturebetween room temperature and 100 C.

6. A process according to claim 1 wherein the reaction of2-methyl-3-hydroxypyridine-4,S-dicarboxaldehyde with a compound of thegeneral formula:

R -NHCH CH SH is carried out in a solvent selected from the groupconsisting of water, methanol, ethanol and dioxane, at a temperaturebetween room temperature and 100 C.

7. A process according to claim 1 wherein the reduction of a compound ofthe general formula:

CHO

8. A process according to claim 1 wherein the phosphorylation of acompound of the general formula:

is carried out at a temperature between 40 and C.

9. A process according to claim 1 wherein a mixture of unreactedstarting materials of the first step is recovered and re-used after thecomplementary addition thereto of the fresh starting materials.

10. A process according to claim 1 followed by an isolation step ofpyridoxal-S-phosphate which comprises adding an organic base selectedfrom the group consisting of 4-aminoantipyrine and wnaphthylamine to thereaction mixture containing pyridoxal-S-phosphate to form thecorresponding Schiff base; isolating the Schiif base; decomposing theSchiif base with an alkali metal hydroxide; removing the liberatedorganic base; and then recovering pyridoxal-S-phosphate from theresultant solution.

11. A compound of the formula:

HO CHO on3 \N/ wherein R represents a member selected from the groupconsisting of a cyclohexyl and benzyl group.

12. 2-(3 hydroxy 5 formyl-2-methyl-4-pyridyl)-3- cyclohexylthiazolidine.

13. 2-(3-hydroxy 5-formyl benzylthiazolidine.

14. A compound of the formula:

2 methyl-4-pyridyl)-3- CHZOH CH3 N 3,124,587 3/1969 Schorre 260-2975HENRY R. JILES, Primary Examiner ALAN L. ROTMAN, Assistant Examiner U.S.Cl. X.R. 260297, 307, 347.8, 609, 999

